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Subjects

Abstract

Human pluripotent cell lines hold enormous promise for the development of cell-based therapies. Safety, however, is a crucial prerequisite condition for clinical applications. Numerous groups have attempted to eliminate potentially harmful cells through the use of suicide genes1, but none has quantitatively defined the safety level of transplant therapies. Here, using genome-engineering strategies, we demonstrate the protection of a suicide system from inactivation in dividing cells. We created a transcriptional link between the suicide gene herpes simplex virus thymidine kinase (HSV-TK) and a cell-division gene (CDK1); this combination is designated the safe-cell system. Furthermore, we used a mathematical model to quantify the safety level of the cell therapy as a function of the number of cells that is needed for the therapy and the type of genome editing that is performed. Even with the highly conservative estimates described here, we anticipate that our solution will rapidly accelerate the entry of cell-based medicine into the clinic.

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Contributions

Q.L. designed and conducted most of the experiments, including targeting of mouse and human ES cells, teratoma analysis, mammary gland tumour analysis, neural in vitro differentiation, mutation rate calculation, as well as analysing the data and writing the manuscript. C.M. designed and conducted experiments, designed and constructed the CDK1–TK targeting cassette, targeted mouse and human ES cells, analysed safe-cell escapees, performed in vitro differentiation assays, analysed data, composed figures and wrote the manuscript. M.V.S. conducted the Monte Carlo simulations and analysed the data. I.G. performed the mathematical modelling. E.J.N. targeted human ES cells, differentiated RPEs and wrote the manuscript. S.H. performed the eye experiment and analysed the data. H.Y. performed in vitro differentiation into endoderm and analysed the data. C.K. performed in vitro differentiation into mesenchymal stem cells and analysed the data. P.Z. performed Southern blots. C.L. performed animal teratoma experiments. K.N. performed animal teratoma experiments, analysed the data and edited the manuscript. M.M. targeted human ES cells. H.-K.S. analysed teratoma histology. A.N. conceived and supervised the study, designed experiments, analysed the data and wrote the manuscript.

Competing interests

A.N., C.M. and Q.L. are inventors on a patent application covering the SC technology (PCT/CA2016/050256). A.N. is a co-founder and shareholder of panCELLa Inc. C.M. is a senior scientist at panCELLa Inc. The other authors declare no competing interests.

a, Generation of human CA1 CDK1–TK/CDK1 and CDK1–TK/CDK1–TK ES cells. b, Southern blot genotyping of human CA1 CDK1–TK/CDK1 and CDK1–TK/CDK1–TK ES cells. The plasmid concatemers are multiple copies of plasmid integration (including backbone). The ampicillin gene in the backbone contains a ScaI restriction enzyme site, which is consistent with the sizes of the band in Southern blots. c, Haematoxylin and eosin staining of a CDK1–TK/CDK1–TK CA1 ES-cell-derived teratoma. d, The efficiency of teratoma formation in NSG mice using human CA1 ES cells. e, Flow cytometry analysis shows a direct correlation between the number of CDK1–TK alleles and mCherry fluorescence levels.

a, Growth of teratomas derived from mouse heterozygous safe-cell ES cells (C2 Cdk1–TK/Cdk1) b, Adult mouse with stabilized subcutaneous tissue (safe-cell ES-cell-derived dormant teratoma), 2.5 months after GCV treatment. c, Growth of teratomas derived from mouse homozygous safe-cell ES cells (C2 Cdk1–TK/Cdk1–TK). d, Growth of teratomas derived from human heterozygous safe-cell ES cells (H1 CDK1–TK/CDK1, clone Exc16); daily GCV treatment. e, Examples of teratomas from human heterozygous safe-cell ES cells showing cyst formation, images of cystic teratomas at dissection are shown next to the corresponding growth line; daily GCV treatment. The graphs with two lines represent mice that had cells injected into both flanks. The graphs with one line represent mice that had cells injected into one flank. The GCV treatment regime varies among mice because each teratoma behaves differently; we started GCV when the teratoma size started to increase. f, Growth of teratomas derived from human homozygous safe-cell ES cells (H1 CDK1–TK/CDK1–TK), GCV treatment was every other day. Images of cystic teratomas are shown next to the corresponding growth line, cysts were drained after dissection to show the difference in tumour weight due to the fluid present in the tissue. Each graph represents one mouse. a, c, d, f, All replicates of these experiments are shown. Source dataSource data

a, Generation of mouse lines and experimental design. b, Growth of mammary gland tumours derived from mouse Cdk1/Cdk1 and Cdk1–TK/Cdk1 mammary epithelial cells with PBS or GCV treatment. c, Growth of mammary gland tumours derived from mouse CDK1–TK/CDK1–TK mammary epithelial cells with PBS or GCV treatment. b, c, The sample sizes of each group are indicated at the bottom of the graphs.

a, Experimental design: mCherry+ cells were selected by sorting to ensure that the starting cell population did not contain escapees. These cells were plated on six-well plates (200 cells per well, in a total of 36 wells) and allowed to grow to 14 cell doublings (this was estimated by counting cells in sample wells). The 36 cultures were then resuspended to a single-cell suspension and each was plated in a 15-cm plate (4 × 106 cells). One day after plating, selection with GCV was started and maintained until escapee colonies appeared. b, Escapee numbers obtained in 36 independent cultures growing from Cdk1–TK/Cdk1 and Cdk1–TK/Cdk1–TK ES cells. c, PCR to determine the presence of TK. d, TaqMan copy number qPCR analysis of Akap7, Sim1 and Cdk1 junction of exon 8 and 3′ UTR, Neurod, Cdk1, TK transgene and Abca on mouse chromosome 10. Data are the copy number calculated by CopyCaller Software v.2.1 and the error bars indicate the range from the minimum to the maximum number. n = 3. The same colour in the background of c and d indicates that they are from the same independent culture. n.d., not determined. e, qPCR to compare TK expression level in Cdk1–TK/Cdk1 escapee clone 2A and C2 wild-type ES cells. Data are mean ± s.e.m., n = 3. f, Summary of the copy number analysis of mouse Cdk1–TK/Cdk1 escapees. a, b, Experiments were repeated twice on a smaller scale but with similar results. d, Experiments were repeated twice with similar results.
Source data

a, The drop of SCL due to aliquoting from a pool of cells relative to non-aliquoted batches of the same size. b, Schematics of the alleles in the CDK1–TK/CDK1–TK human ES cells used in the quality control. c, Workflow schematic of performing quality control (QC) on several ES cell batches. d, An example of the flow cytometry for the quality control of nine clonally derived batches. e, An example of PCR for the quality control of nine clonally derived batches.

a, b, Fundoscopy, optical coherence tomography and fluorescence imaging of eyes transplanted with safe-cell RPE and safe-cell ES cells (four-week GCV treatment). The absence of a mCherry signal indicates that ES cell growth has not occurred. b, Bottom, images of the green fluorescence channel are included to illustrate that the observed signal in the red fluorescence channel is actually autofluorescence. This experiment was repeated four times in four mice with similar results. c, Histological analysis of the eye presented in d. d, Fundoscopy, optical coherence tomography and fluorescence imaging of eyes transplanted with safe-cell RPE and safe-cell ES cells (PBS treatment). This experiment was repeated twice in two mice with similar results. e, f, Fundoscopy, optical coherence tomography and fluorescence imaging of eyes transplanted with safe-cell RPE and safe-cell ES cells, mCherry signal is detectable and indicates ES cell growth. GCV treatment began three weeks post-injection following an initial PBS treatment. This experiment was repeated four times in three mice with similar results. g, Fundoscopy, optical coherence tomography and fluorescence imaging of eyes receiving only safe-cell RPE cells (four-week GCV treatment). This demonstrates that GCV treatment did not affect the RPE cells. This experiment was repeated five times in three mice with similar results. h, Fundoscopy, optical coherence tomography and fluorescence imaging of eyes receiving only safe-cell RPE cells (four-week PBS treatment). This experiment was repeated six times in three mice with similar results. i, Fundoscopy, optical coherence tomography and fluorescence imaging of eyes receiving only HAMC (four-week GCV treatment). This experiment was repeated twice in one mouse with similar results.

This file contains Supplementary Table 1. To generate a list of CDLCDEL candidates, we cross-referenced genes whose knock-out has an early embryonic lethal phenotype in mice (www.informatics.jax.org/phenotypes.shtml) with data from a genome-wide CRISPR/Cas9 mutagenesis screen for essential genes in human cancer cell lines41. We found 167 genes with a high-fitness score (< -1.0) that also have a known early-embryonic lethal phenotype